EP3476865B1 - Procédé de construction d'un modèle animal humanisé modifié par le gène pd-1 et son utilisation - Google Patents

Procédé de construction d'un modèle animal humanisé modifié par le gène pd-1 et son utilisation Download PDF

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EP3476865B1
EP3476865B1 EP17819238.1A EP17819238A EP3476865B1 EP 3476865 B1 EP3476865 B1 EP 3476865B1 EP 17819238 A EP17819238 A EP 17819238A EP 3476865 B1 EP3476865 B1 EP 3476865B1
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mouse
gene
mice
human
humanized
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EP3476865A4 (fr
EP3476865A1 (fr
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Yuelei SHEN
yang BAI
Rui Huang
Xiaofei Zhou
Yuting HU
Yanan GUO
Jichao DU
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Biocytogen Pharmaceuticals Beijing Co Ltd
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Biocytogen Pharmaceuticals Beijing Co Ltd
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Definitions

  • the present application relates to the field of mouse genetic engineering and genetic modification, and in particular, to a method for constructing a PD-1 gene-modified humanized mouse model based on the CRISPR/Cas9 technique and a use thereof in biomedicine.
  • a humanized animal model refers to an animal model that carries human functional genes, cells or tissues. This model is typically used as an alternative living model for research on human diseases, and has tremendous advantages and a prospect of extensive applications in understanding pathogenesis and drug screening.
  • mice have been one of the mostly used biological models. Considering the differences between mice and humans in physiological, pathological, and many other aspects, however, it is particularly important to construct humanized mouse models that carry human functional genes, cells or tissues.
  • one method is to use humanized genetic animal models that are prepared by "placing" human genes on chromosomes of rats and mice using a method of gene modification.
  • tumor immunotherapy is one of the most promising research directions in the field of tumor treatment.
  • the journal Science ranked tumor immunotherapy as No. 1 of Top Ten Science Breakthroughs in 2013.
  • studies on PD-1/PD-L1 channel inhibitors have attracted particular attention.
  • PD-1 programmed death-1
  • PD-L1 and PD-L2 Two ligands of PD-1 (PD-L1 and PD-L2) are extensively expressed in antigen-presenting cells (APCs) and others.
  • APCs antigen-presenting cells
  • the interaction between PD-1 and its receptors plays an important role in negative regulation of immune response.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the micro-environment of tumor sites can induce an expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is favorable for genesis and growth of tumors, induces the apoptosis of anti-tumor T cells, and then evades the attack from an immune system.
  • tumor cells By inhibiting the binding of PD-1 and its ligands, tumor cells can be exposed to the attack of an immune system, and then the effect of killing tumor tissues and treating cancers can be achieved.
  • NCI National Cancer Institute
  • Nihimura et al. prepared BALB/c mice with PD-1 knocked out in 2001. These models were mainly used in studies on functions of the PD-1 gene and relevant disease mechanisms.
  • WO 2015/196051 A1 describes non-human animals having a humanized PD-1 gene and expressing a PD-1 polypeptide that includes a human portion and an endogenous portion.
  • US 2016/0157469 A1 describes non-human animals having a genetic modification to an endogenous CD274 gene so that the animals express a PD-L1 polypeptide that includes a human portion and an endogenous portion.
  • the present invention is hereby provided in light of the insufficiencies and defects of the prior art, so as to make efficacy trials in early stage clinical trials more effective and improve the success rate of research and development.
  • An object of the present invention is to provide a PD-1 gene-modified humanized non-human mouse model.
  • a second object of the present invention is to provide a method for preparing the PD-1 gene-modified humanized mouse model.
  • a third object of the present invention is to provide an application of the model.
  • the present disclosure provides a PD-1 gene-modified humanized mouse model, the genome thereof contains human PD-1 gene fragments and can normally express PD-1 functional protein, and the expressed protein contains a functional domain of human PD-1 genes.
  • the PD-1 gene modification is a gene modification performed on the second exon of the PD-1 gene.
  • the background of the mouse is C57BL/6.
  • nucleotides 11053-11385 of its PD-1 gene are replaced by a human DNA fragment, and other mouse PD-1 regions including the transmembrane region are retained; the human DNA fragment is as shown by SEQ ID NO: 21.
  • nucleotides 11053-11385 of its PD-1 gene are replaced by a human DNA fragment, and 14/13bp of the left and right edges of the second exon and other mouse PD-1 regions including the transmembrane region are retained; the human DNA fragment is as shown by SEQ ID NO: 21.
  • the present disclosure provides the DNA sequence of a humanized mouse PD-1 gene, and the protein encoded with the DNA sequence contains functional domains of human PD-1 proteins.
  • the present disclosure provides the DNA sequence of a humanized mouse PD-1 gene, wherein nucleotides 11053-11385 of a mouse PD-1 gene are replaced by a human DNA fragment, and 14/13bp of each of the left and right edges of the second exon and other mouse PD-1 regions including the transmembrane region are retained;
  • the human DNA fragment is as shown by SEQ ID NO: 21 or is a DNA fragment having 80% or higher homology with this fragment;
  • the DNA sequence of the humanized mouse PD-1 gene contains a nucleotide sequence shown by SEQ ID NO: 14 or a DNA fragment having 80% or higher homology with the DNA sequence of the humanized mouse PD-1 gene.
  • the upstream and downstream primer sequences used for amplifying the DNA sequence of the humanized mouse PD-1 gene are as shown by SEQ ID NO: 22-23.
  • DNA sequence of the humanized mouse PD-1 gene its CDS sequence is shown by SEQ ID NO: 15 or a DNA fragment having 80% or higher homology with SEQ ID NO: 15
  • its mRNA sequence is shown by SEQ ID NO: 16 or a DNA fragment having 80% or higher homology with SEQ ID NO: 16
  • the protein sequence encoded thereby is shown by SEQ ID NO: 17 or a specific amino acid fragment thereof with unchanged functions.
  • the present disclosure provides a use of the DNA sequence of a humanized mouse PD-1 gene in constructing the PD-1 gene-modified humanized animal model, wherein nucleotides 11053-11385 of the second exon of the mouse PD-1 gene are replaced by a human DNA fragment, and other mouse PD-1 regions including the transmembrane region are retained, the human DNA fragment is as shown by SEQ ID NO: 21, and the DNA sequence of the humanized mouse PD-1 gene contains a nucleotide sequence shown by SEQ ID NO: 14.
  • nucleotides 11053-11385 of the second exon of the mouse PD-1 gene are replaced by a human DNA fragment, and 14/13bp is retained for each of the left and right edges.
  • the use is to inject an in vitro transcription product containing an expression vector of the DNA sequence of the humanized mouse PD-1 gene and Cas9mRNA, and sgRNA plasmid of the second exon of the mouse PD-1 gene into a cytoplasm or nucleus of a mouse fertilized egg, and to transplant the fertilized egg into a receiving female mouse for producing the PD-1 gene-modified humanized mouse model.
  • the present disclosure provides sgRNA of the second exon of the specifically targeted mouse PD-1 gene, and its target site sequence is as shown by SEQ ID NO: 3 or SEQ ID NO: 8.
  • the upstream and downstream single stranded sequences as shown by SEQ ID NO: 9-10, respectively, of a target site sequence as shown by SEQ ID NO: 3 are synthesized; the upstream and downstream single stranded sequences, as shown by SEQ ID NO: 11-12, respectively, of a target site sequence as shown by SEQ ID NO: 8 are synthesized.
  • the CRISPR/Cas9 targeting vector that contains the DNA sequence of the sgRNA falls within the protection scope of the present disclosure.
  • the present disclosure provides a use of the sgRNA or the CRISPR/Cas9 targeting vector in preparing a PD-1 gene-modified humanized mouse model.
  • a method for preparing a PD-1 gene-modified humanized animal model comprising the following steps:
  • the DNA of a wild-type C57BL/6 mouse genome is used as a template for PCR amplification of the 5' homology arm fragment and the 3' homology arm fragment, and the amplified primer pair sequences are as shown by SEQ ID NO: 19-20 and SEQ ID NO: 25-26, respectively; the plasmid is PV-4G.
  • the plasmid of sgRNA of the second exon of the mouse PD-1 gene from the step (2) is obtained by connecting the sgRNA to a pT7-sgRNA plasmid.
  • the present disclosure further provides a PD-1 gene-modified humanized animal model prepared using the above method.
  • the present disclosure provides a use of the animal model, the DNA sequence of the humanized mouse PD-1 gene, the sgRNA for the second exon of the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector in preparing PD-1 or PD-L1 regulators or drugs.
  • the present disclosure provides a use of the animal model, the DNA sequence of the humanized mouse PD-1 gene, the sgRNA for the second exon of the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector in research on PD-1 or PD-L1 signal mechanism.
  • the present disclosure provides a use of the animal model, the DNA sequence of the humanized mouse PD-1 gene, the sgRNA for the second exon of the mouse PD-1 gene, or its CRISPR/Cas9 targeting vector in oncological research.
  • the present disclosure further relates to sperms, eggs, fertilized eggs, embryos, progenies, tissues or cells from a gene-modified animal, research on PD-1/PD-L1 signal mechanism of the above biological material, and use in pre-clinical experiments, such as research on drug toxicity of PD-1/PD-L1 regulators and screening and development of PD-1/PD-L1 regulators.
  • the present disclosure provides a use of the animal model, the DNA sequence of the humanized mouse PD-1 gene, the sgRNA, or CRISPR/Cas9 targeting vector comprising the same in R&D of PD-1 or PD-L1 regulators or drugs, in research on PD-1 or PD-L1 signaling mechanism, and in oncological research.
  • the present disclosure provides a mouse carrying humanized and modified PD-1 genes, wherein its genome contains human PD-1 gene fragments, can normally express PD-1 functional protein, and the expressed protein contains a functional domain of human PD-1 genes.
  • nucleotides 11053-11385 of the second exon of its PD-1 gene are replaced by a DNA fragment of human PD-1 gene, 14/13bp is retained for each of the left and right edges, and other mouse PD-1 regions including the transmembrane region are retained; and the human DNA fragment is as shown by SEQ ID NO: 21.
  • the present disclosure provides a use of the mouse model or the mouse carrying humanized and modified PD-1 genes in preparing a multi-gene humanized animal model.
  • the multi-gene humanized animal model prepared using the mouse model or the mouse carrying humanized and modified PD-1 genes through copulation or further with a gene editing method also falls within the protection scope of the present disclosure.
  • the drugs are monoclonal antibody, bispecific antibody, or polyclonal antibody drugs, as well as other biological or chemical drugs.
  • the present disclosure further provides a cell or tissue isolated from the mouse model or any mouse.
  • the present disclosure further provides a use of the mouse, cell, or tissue in R&D of PD-1 or PD-L1 regulators or drugs, in research on PD-1 or PD-L1 signaling mechanism, and in oncological research.
  • the present disclosure further provides a use of the mouse, cell, or tissue in evaluating effectiveness of targeted PD-1/PD-L1 drugs or in screening targeted PD-1/PD-L1 drugs.
  • the present disclosure further provides a use of the mouse, cell, or tissue in evaluating effectiveness of joint administration of targeted PD-1/PD-L1 drugs and other drugs or in screening combined drugs for joint administration of targeted PD-1/PD-L1 drugs and other drugs.
  • the present disclosure further provides a use of mouse, cell, or tissue in screening regulators for human PD-1/PD-L1 signal pathway.
  • the present disclosure further provides a method for evaluating effectiveness of targeted PD-1/PD-L1 drugs, which uses the mouse, cell, or tissue to evaluate drugs
  • the present disclosure further provides a method for evaluating effectiveness of joint administration of drugs targeting PD-1/PD-L1 combined with other drugs, which uses the mouse, cell, or tissue to evaluate combined drugs for joint administration of drugs targeting PD-1/PD-L1 combined with other drugs.
  • the present disclosure further provides a method for screening drugs targeting PD-1/PD-L1, which uses the mouse, cell, or tissue to screen drugs
  • the present disclosure further provides a method for screening joint administration of drugs targeting PD-1/PD-L1 combined with other drugs, which uses the mouse, cell, or tissue to screen combined drugs for joint administration of drugs targeting PD-1/PD-L1 combined with other drugs.
  • the drugs targeting PD-1/PD-L1 are drugs for treating tumors.
  • the drugs are monoclonal antibody, bispecific antibody, or polyclonal antibody drugs, as well as other biological or chemical drugs.
  • the present disclosure has the following features: 1) for the mouse model according to the present disclosure, the second exon of its PD-1 gene is partially and manually replaced by the second exon of human PD-1 gene in a manner of DNA sequence homologous recombination; 2) the animal model according to the present disclosure can express a PD-1 protein containing the functional domain of the human PD-1 protein in the animal body.
  • the adoption of this method can minimize the impact on humanization caused by gene splicing and editing; and retain other mouse PD-1 gene functional regions including the transmembrane region, so as to avoid abnormality in mouse expression of the PD-1 protein and physiological functions related to the PD-1 protein as a result of gene humanization.
  • Biochemical reagents used in examples of the present application include EcoRI, EcoRV, HindIII, KpnI, BglII enzymes purchased from NEB with the article numbers thereof being R3101M, R3195M, R3104M, R3142M, and R0144M, respectively; NucleoBond ® Xtra Maxi Plus EF purchased from Macherey-Nagel with the article number being 740426; Kanamycin purchased from Amresco with the article number being 0408; Ambion in vitro transcription kit purchased from Ambion with the article number being AM1354; AIO and UCA kits purchased from Biocytogen with the article numbers being BCG-DX-004 and BCG-DX-001, respectively; Cas9mRNA was from SIGMA with the article number being CAS9MRNA-1EA; mouse colon cancer cells MC38 purchased from EK-Bioscience; E-coli TOP10 competent cells purchased from Tiangen with the article number being CB104-02; anti-mouse PD-1 from Biolegend
  • the target sequence determines the targeting specificity of small guide RNA (sgRNA) and the efficiency of Cas9 cleavage at the target gene. Therefore, target sequence selection was important for sgRNA vector construction.
  • sgRNA small guide RNA
  • the UCA kit was used to detect activities of a plurality of guide sgRNAs screened from Example 1, and it can be seen from the results that the guide sgRNAs have different activities. See FIG. 1 for the detection results.
  • sgRNA3 and sgRNA8 were selected therefrom for subsequent experiments.
  • Upstream and downstream single strands of the sgRNAs were synthesized. See Table 1: Table 1 List of sgRNA3 and sgRNA8 sequences sgRNA3 sequence SEQ ID NO: 9 Upstream: 5'-tagaaggtgagggacctcc-3' SEQ ID NO: 10 Downstream: 5'-ggaggtccctcaccttcta-3' sgRNA8 sequence SEQ ID NO: 11 Upstream: 5'-caaaaatcgaggagagccc-3' SEQ ID NO: 12 Downstream: 5'-gggctctcctcgatttttg-3'
  • the source of pT7-sgRNA plasmids see FIG. 2 for the pT7-sgRNA vector map.
  • the plasmid backbone was from Takara with the Catalog No. 3299.
  • DNA fragments containing a T7 promoter (taatacgactcactatagg) and sgRNA scaffold were synthesized by a plasmid synthesis company (see SEQ ID NO: 13) and were connected to the skeleton vector through enzyme digestion (EcoRI and BamHI). As verified through sequencing by a professional sequencing company, the results show that the target plasmids were obtained.
  • TAGG was added to the 5' ends of the upstream single strands and AAAC was added to the 5' ends of the downstream single strands.
  • AAAC was added to the 5' ends of the downstream single strands.
  • the upstream and downstream single strands were connected to the pT7-sgRNA plasmids (the plasmid was linearized first using BbsI), respectively, to obtain the expression vectors pT7-PD-3 and pT7-PD-8.
  • connection reaction system sgRNA annealing product 1 ⁇ L (0.5 ⁇ M) pT7-sgRNA vector 1 ⁇ L (10 ng) T4 DNA Ligase 1 ⁇ L (5 U) 10xT4 DNA Ligase buffer 1 ⁇ L 50% PEG4000 1 ⁇ L H 2 O Add to 10 ⁇ L
  • reaction conditions were as follows: connect at room temperature for 10 to 30 min, transfer into 30 ⁇ L TOP10 competent cells, then take 200 ⁇ L for coating onto a Kan resistant plate, culture at 37°C for at least 12 h, select 2 clones for inoculation into an LB medium having Kan resistance (5 ml), and shake to culture at 37°C and 250 rpm for at least 12 h.
  • Nucleotides 11053-11385 of the second exon of the mouse PD-1 gene were replaced by a human DNA fragment (SEQ ID NO: 21), and the ultimately obtained DNA sequence of the modified humanized mouse PD-1 (chimeric PD-1 DNA) was shown by SEQ ID NO: 14.
  • SEQ ID NO: 14 only lists the DNA sequence of the modified portion, wherein the 219-551 bp region was a human fragment.
  • a human DNA fragment was replaced onto the second exon of the mouse PD-1 gene to ultimately obtain a humanized mouse.
  • the CDS sequence and mRNA sequence of the humanized mouse PD-1 were shown by SEQ ID NO: 15 and SEQ ID NO: 16, respectively; for SEQ ID NO: 15 (869 bp), the sites 91-423 were a human fragment, and for SEQ ID NO: 16 (1972 bp), the sites 154-486 were a human fragment; the human/mouse chimeric PD-1 protein sequence was as shown by SEQ ID NO: 17, wherein the 31-141 bp region was a human fragment.
  • upstream primers of 3 homologous recombinant fragments, matching downstream primers, and relevant sequences were designed. Specifically, the DNA of a wild-type C57BL/6 mouse genome was used as a template for PCR amplification to obtain a5' homology arm fragment and a 3' homology arm fragment; the DNA of a human genome was used as a template for PCR amplification to obtain a human DNA fragment.
  • the 5' homology arm (1770 bp) the nucleotide of the site 94041502-94043271 (SEQ ID NO: 18) with an NCBI login ID being NC_000067.6, the upstream primer (SEQ ID NO:19), and the downstream primer (SEQ ID NO:20).
  • the human DNA fragment (333 bp) the nucleotide of the site 241852634-241852966 (SEQ ID NO:21) with an NCBI login ID being NC_000002.12, the upstream primer (SEQ ID NO:22), and the downstream primer (SEQ ID NO:23).
  • the 5' homology arm fragment, the 3' homology arm fragment, and the human DNA fragment were connected, via the AIO kit, to the TV-4G plasmid provided with the kit, to ultimately obtain the vector TV-PD.
  • Fertilized egg of C57BL/6 mice were taken, a microinjector was used to inject the in vitro transcription product of pT7-PD-3 and pT7-PD-8 plasmids prepared in Example 4 (using the Ambion in vitro transcription kit for transcription according to the instructions), Cas9mRNA and TV-PD plasmids that were premixed into a cytoplasm or nucleus of a mouse fertilized egg.
  • Microinjection of embryos was performed according to the method in the "Mouse Embryo Operation Experiment Manual (3 rd Ed)," and the fertilized egg after injection were transferred into a culture medium for a short period of culture.
  • mice having the C57BL/6 background (i.e., the Founder mice that were the F0 generation) were obtained.
  • the obtained mice were subjected to hybridization and auto-copulation to increase the population and establish a stable mouse breed.
  • the immune node humanized mice obtained using this method were named as B-hPD-1.
  • L-GT-F was at the left side of the 5' homology arm
  • R-GT-R was at the right side of the 3' homology arm
  • R-GT-F and L-GT-R were both on the second exon.
  • the 5' end upstream primer L-GT-F (SEQ ID NO:27), and the downstream primer L-GT-R (SEQ ID NO:28).
  • the 3' end upstream primer R-GT-F (SEQ ID NO:29), and the downstream primer R-GT-R (SEQ ID NO:30).
  • the PCR reaction system (20 ⁇ L) was shown in Table 3: Table 3 10x buffer 2 ⁇ L dNTP (2 mM) 2 ⁇ L MgSO 4 (25 mM) 0.8 ⁇ L Upstream primer (10 ⁇ M) 0.6 ⁇ L Downstream primer (10 ⁇ M) 0.6 ⁇ L Tail genome DNA 200 ng KOD-Plus-(1 U/ ⁇ L) 0.6 ⁇ L
  • the F0 generation mice identified to be positive were subjected to copulation with wild-type mice to obtain F1 generation mice.
  • PCR analysis was performed on the tail genome DNA of the F1 generation mice.
  • the PCR system, reaction conditions, and primers re the same as those for the F0 generation genotyping. See FIG. 5 for PCR results of the F1 generation mice, which indicates that 6 F1 generation mice were positive mice with specific numbers of F1-1, F1-2, F1-3, F1-5, F1-7, and F1-8.
  • the Southern blot method was used to determine whether there was random insertion in the 6 mice determined by PCR to be positive (F1-1, F1-2, F1-3, F1-5, F1-7, and F1-8).
  • the genomic DNA was extracted from the mouse tail KpnI and BglII enzymes were selected to digest the genomes, respectively, the digestion products were transferred to membrane and hybridized.
  • the probes P1 and P2 were on the outside of the 3' homology arm and the humanized fragment, respectively.
  • the probe synthesis primers were P1-F (SEQ ID NO:31), P1-R (SEQ ID NO:32); P2-F (SEQ ID NO:33), P2-R (SEQ ID NO:34), respectively.
  • mice that have been successfully prepared produce, via probe hybridization, bands of 2.7 kb or 3.7 kb, respectively, while the wild-type C57BL/6 mouse genome only has bands of 7.3 kb or 3.7 kb with no production of hybrid bands.
  • the F1 generation mice obtained in Example 8 were taken, their T lymphocytes (CD3+) were taken, which were subjected to flow cytometry with anti-mouse PD-1 and anti-human PD-1 antibodies. From the flow cytometry results ( FIG. 7 ), it can be seen that the percent of the double-positive cells obtained by staining the two antibodies anti-human PD-1 and anti-mouse CD3 was 92%, indicating that the PD-1 gene-modified humanized mouse obtained using this method can express humanized PD-1 proteins.
  • F1 generation mice obtained in Example 8 were subjected to mutual copulation to obtain F2 generation PD-1 gene humanized homozygotes.
  • One PD-1 gene humanized homozygote (8 week old) was selected, one wild-type C57BL/6 mouse was taken as a control, 7.5 ⁇ g mouse CD3 antibody was administered to the mice through intraperitoneal injection, and after 24h, the mice were subjected to euthanasia through neck break. The spleens were collected and grinded. The ground samples were then passed through 70 ⁇ m cell mesh, the filtered cell suspensions were centrifuged and the supernatants were discarded; the erythrocyte lysis solution was added for lysis of 5 min, and then PBS solution was added to neutralize the lysis reaction. The solution was centrifuged again and the supernatants were discarded. The cells were washed once with PBS, then subjected to FACS detection and RT-PCR detection, respectively.
  • the FACS detection the anti-mouse PD-1 antibody mPD-1 PE and the anti-mouse T-cell surface antibody mTcR ⁇ , as well as the anti-human PD-1 antibody hPD-1 FITC and the anti-mouse T-cell surface antibody mTcR ⁇ , were used simultaneously for staining T-cell extracellular proteins, and after the cells were cleaned with PBS, flow cytometry was performed to detect protection expression.
  • the flow cytometry results FIG.
  • RNA of spleen cells was extracted from wild-type C57BL/6 mice and B-hPD-1 homozygous mice, and a reverse transcription kit was used for reverse transcription to cDNA,
  • the PCR reaction system was 20 ⁇ L, and the reaction conditions were as follows: 95°C, 5 min; (95°C, 30 sec; 60°C, 30 sec; 72°C, 30 sec, 35 cycles); 72°C, 10 min; keeping temperature constant at 4°C. GAPDH was used as an internal control.
  • mice The experimental results show ( FIG. 9 ) that mRNA expression of the mouse PD-1 can be detected in the activated cells of wild-type C57BL/6 mice, and mRNA expression of the humanized PD-1 can be detected in the activated cells of B-hPD-1 homozygous mice.
  • mice B-hPD-1 homozygous mice (4-6 weeks old) were taken, mouse colon cancer cells MC38 or modified MC38-hPDL1 (i.e., PD-L1 gene humanized MC38 cells) were subcutaneously inoculated.
  • the tumor volume was about 100 mm 3
  • the mice were randomly divided into a control group or a treatment group.
  • one of the above 2 antibodies was randomly selected and administered at different dosages (0.3-25 mg/kg), and the same volume of a blank solvent was injected for the control group.
  • the tumor volume was measured twice a week and the body weight were measured for each mouse.
  • euthanasia was performed when the tumor volume of a single mouse reached 3000 mm 3 .
  • the specific experiment scheme was as follows:
  • the animals have good health conditions in the experiments of all groups. At the end of all experiments, all the animals in all groups have gained weight. There was no significant difference in weight throughout the entire experiment period between all the treatment groups and control groups ( FIGs. 10 , 12 , 14 , and 16 ). In terms of the tumor measurements ( FIGs. 11 , 13 , 15 , and 17 ), however, tumors were growing continuously in the experiment period for all mice in the control groups. Compared with the control groups thereof, all the treatment groups have the volume of tumors shrunk to different degrees and/or disappears. Therefore, the treatment by either of the two drugs for human PD-1/PD-L1 signal channels on the market has significantly inhibited the growth of tumors in the mice.
  • mice in the treatment group have a tumor volume obviously smaller than that of the control group, and the difference was significant (p ⁇ 0.05), and TGI TV was 102%. Therefore, it was proven that according to the administration manner, the anti-human PD-1 antibody Keytruda has a significant inhibitory effort on tumors inside B-hPD-1 mice (TGI TV > 60%), has good capabilities of treating and inhibiting tumor growth, does not have obvious toxicity on the animals, and has good safety.
  • the average tumor volume of the control group was 1589 ⁇ 652 mm 3
  • the average tumor volume of the treatment group was 223 ⁇ 270 mm 3 , 277 ⁇ 397 mm 3 , 201 ⁇ 186 mm 3 , and 437 ⁇ 515 mm 3 at doses of 10 mg/kg, 3 mg/kg, 1 mg/kg, and 0.3 mg/kg, respectively.
  • All mice in the treatment groups have a tumor volume obviously smaller than that of the control group, and the difference was significant (p ⁇ 0.05) compared with the tumor volume of the control group.
  • the optimal therapeutic effect was achieved after 3 weeks of administration. After the administration was stopped, obvious tumor growth occurs in the treatment groups having low dosage.
  • TGI TV was 92.4%, 89.0%, 94.0%, and 78.2%, respectively, indicating that the anti-human PD-1 antibody Keytruda has a significant inhibitory effort on tumors (TGI TV > 60%) and shows different therapeutic effects inside B-hPD-1 mice at different dosages.
  • mice in the treatment group have a tumor volume obviously smaller than that of the control group, and TGI TV was 79.7%. Therefore, it was proven that according to the administration manner, the anti-human PD-1 antibody Tecentriq has a significant inhibitory effort on tumors inside B-hPD-1 mice (TGI TV > 60%), has strong capabilities of inhibiting tumor growth, does not have obvious toxicity on the animals, and has good safety.
  • the average tumor volume of the control group was 2464 ⁇ 1914 mm 3
  • the average tumor volume of the treatment groups was 219 ⁇ 326 mm 3 , 1028 ⁇ 963 mm 3 , and 1044 ⁇ 432 mm 3 at doses of 10 mg/kg, 3 mg/kg, and 1 mg/kg, respectively.
  • All mice in the treatment groups have a tumor volume obviously smaller than that of the control group.
  • TGI TV was 97.2%, 93.7%, and 61.2%, respectively, indicating that the anti-human PD-1 antibody Tecentriq has a significant inhibitory effort on tumors (TGI TV > 60%) at different dosages, and higher doses have better therapeutic effects.
  • mice B-hPD-1 homozygous mice (4-6 weeks old) were taken, 5 ⁇ 10 5 mouse colon cancer cells MC38-hPDL1 were subcutaneously inoculated at right side (the same as Example 10).
  • the tumor volume was about 100 mm 3
  • the dosages were 0.1-10 mg/kg, and a blank solvent was injected for the control group.
  • the tumor volume was measured and the mice were weighed twice a week.
  • euthanasia was performed when the tumor volume of a single mouse reached 3000 mm 3 .
  • Table 9 The specific administration or administration combination, dosage, administration manner and frequency were listed in Table 9.
  • mice in the control group (G1) and the treatment groups using antibodies (G3 and G5) survive at the experiment end-point.
  • the average tumor volume of the control group (G1) was 1716 ⁇ 789 mm 3
  • the average tumor volume of the treatment group with Cisplatin only (G2) was 612 ⁇ 510 mm 3
  • the average tumor volume of the treatment group with Keytruda only (G3) was 1267 ⁇ 619 mm 3
  • the average tumor volume of the treatment group of Cisplatin combined with Keytruda (G4) was 496 ⁇ 160 mm 3
  • the average tumor volume of the treatment group with Tecentriq only (G5) was 1234 ⁇ 977 mm 3
  • the average tumor volume of the treatment group of Cisplatin combined with Tecentriq (G6) was 427 ⁇ 148 mm 3 .
  • the tumor volume of the mice in the treatment group with combination of Cisplatin and Keytruda was significantly smaller than that of Cisplatin only (G2) or Keytruda only (G3); the tumor volume of the mice in the treatment group with combination of Cisplatin and Tecentriq (G6) was significantly smaller than that of Cisplatin only (G2) or Tecentriq only (G5).
  • the experimental results show that the chemotherapy drug Cisplatin has certain tumor inhibitory effect, but has certain toxicity that can lead to mouse death.
  • the TGI TV value also shows that the therapeutic effect of joint administration of the above two monoclonal antibodies and the chemotherapy drug Cisplatin was superior to the therapeutic effect of separate administration of the monoclonal antibodies or the chemotherapy drug.
  • B-hPD-1 mouse model was respond to existing anti-human PD-1/PD-L1 inhibitors and chemotherapy drug or combination thereof, and has shown dosage correlation with tumor growth inhibition.
  • the following Examples were selected anti-human PD-1/PD-L1 inhibitors to further prove that B-hPD-1 mice can be used as alternative living model for in vivo research and for screening, evaluating and treating of human PD-1/PD-L1 pathway regulators.
  • mice B-hPD-1 homozygous mice (4-6 weeks old) were taken, 5 ⁇ 10 5 mouse colon cancer cells MC38-hPDL1 were subcutaneously inoculated at right side (the same as Example 10).
  • the tumor volume was about 100 mm 3
  • the positive control Tecentriq or one of two anti-human PD-L1 antibodies was randomly selected, all dosages were 3 mg/kg, and a blank solvent was injected for the control group.
  • the administration manner was intraperitoneal injection, once per 2 days for a total of 8 times.
  • the tumor volume was measured twice a week.
  • euthanasia was performed when the tumor volume of a single mouse reached 3000 mm 3 .
  • Table 11 Main data and analytical results of all experiments were listed in Table 11, specifically comprising tumor volumes at the time of group division and at 10 days after group division, tumor volumes when the experiments end, situation of mice survival, situation of tumor-free mice, Tumor Growth Inhibition Value (TGIrv), and statistical difference (P values) in mouse weights and tumor volumes between mice in the treatment groups and the control group.
  • TGIrv Tumor Growth Inhibition Value
  • P values statistical difference
  • Table 11 Tumor volume (mm 3 ) Survival situation Tumor free situation TGI TV % P values Day 10 Day 20 Day 27 Weight Tumor volume Control group G1 111 ⁇ 27 247 ⁇ 80 409 ⁇ 131 5/5 0/5 N/A N/A N/A Treat ment grou ps G2(Tecentriq) 111 ⁇ 25 60 ⁇ 70 75 ⁇ 113 5/5 0/5 112.2 0.610 0.003 G3(PDL1-Ab1) 111 ⁇ 29 89 ⁇ 54 101 ⁇ 128 5/5 2/5 103.3 0.899 0.006 G4(PDL1-Ab2) 112 ⁇ 25 180 ⁇ 71 232 ⁇ 88 5/5 0/5 59.8 0.652 0.036
  • the average tumor volume of the control group (G1) was 409 ⁇ 131 mm 3
  • the average tumor volume of the Tecentriq treatment group (G2) was 75 ⁇ 113 mm 3
  • the average tumor volume of the PDL1-Ab1 antibody treatment group (G3) was 101 ⁇ 128 mm 3
  • the average tumor volume of the PDL1-Ab2 antibody treatment group (G4) was 232 ⁇ 88 mm 3 .
  • the difference in tumor volume was not significant between mice in the PDL1-Ab1 antibody treatment group (G3) and the Tecentriq treatment group (G2), while there was a significant difference in tumor volume between G2, G3 and the control group (G1) (P ⁇ 0.05), TGI TV was 112.2% and 103.3%, respectively.
  • the tumor volume of the mice in the PDL1-Ab2 antibody treatment group (G4) was significantly bigger than that of the Tecentriq treatment group (G2) and the PDL1-Ab1 antibody treatment group (G3), indicating that the anti-human PDL1-Ab1 antibody has similar efficacy as that of the positive control Tecentriq under the same dosage and frequency, leading to equivalent effect on inhibiting tumor growth, while the efficacy of the anti-human PDL1-Ab2 antibody was not as good as that of Tecentriq or the PDL1-Ab1 antibody.
  • This experiment proves that the B-hPD-1 mice can be used for screening drugs (e.g. antibodies) targeting human PD-L1 and for in vivo efficacy detection.
  • mice B-hPD-1 homozygous mice (4-6 weeks old) were taken, mouse colon cancer cells MC38 (5 ⁇ 10 5 ) were subcutaneously inoculated.
  • the tumor volume was about 100 mm 3
  • the treatment group one of five anti-human PD-1 antibodies was randomly selected, all dosages were 10 mg/kg, and a blank solvent was injected for the control group.
  • the administration manner was intraperitoneal injection, once per 3 days for a total of 6 times.
  • the tumor volume was measured twice a week.
  • euthanasia was performed when the tumor volume of a single mouse reached 3000 mm 3 .
  • Table 12 Main data and analytical results of all experiments were listed in Table 12, specifically comprising tumor volumes before group division and after group division, tumor volumes when the experiments end, situation of mice survival, situation of tumor-free mice, Tumor Growth Inhibition Value (TGIrv), and statistical difference (P values) in mouse weights and tumor volumes between mice in the treatment groups and the control group.
  • TGIrv Tumor Growth Inhibition Value
  • P values statistical difference
  • the B-hPD-1 mice using this method or already prepared can be further used to prepare a double-gene humanized or multi-gene humanized mouse model.
  • the fertilized egg used in the microinjection and embryo transfer were fertilized egg from other gene modified mice.
  • the fertilized egg of the B-hPD-1 mice can be selected for gene editing to further obtain a PD-1 gene humanized and other gene modified double-gene or multi-gene modified or humanized mouse model.
  • the B-hPD-1 homozygous mice or heterozygous mice obtained using this method can copulate with other gene modified homozygous or heterozygous mice, the progenies thereof were screened, and according to the Mendel's genetic law, there was a probability that a PD-1 gene humanized and other gene modified double-gene or multi-gene modified or humanized mouse model can be obtained. Then, the heterozygous mice were subjected to mutual copulation to obtain double-gene or multi-gene modified or humanized heterozygous mice.
  • CTLA-4/PD-1 mice The preparation of double humanized CTLA-4/PD-1 mice was used as an example. Since mouse CTLA-4 gene and PD-1 gene were on the same chromosome (#1 chromosome), the fertilized egg of B-hCTLA-4 (CTLA-4 gene humanized) mice were selected for gene editing during the microinjection in Example 8, and the CTLA-4/PD-1 gene double humanized mice were ultimately obtained through screening positive mouse progenies.
  • mice One double humanized CTLA-4/PD-1 heterozygote (6-weeks old) was selected, two wild-type C57BL/6 mice were selected as the control, 7.5 ⁇ g mouse CD3 antibody was administered to the mice through intraperitoneal injection, and after 28 h, the mice were subjected to euthanasia through neck break. Their spleens were taken, ground and filtered through a 70 ⁇ m cell screen. The filtered cell suspension was centrifuged, the supernatant was discarded, an erythrocyte lysate was added, after 5 min of lysis, and a PBS solution was added to neutralize the lysis reaction.
  • FIGs. 25A, 25B, 25C The solution was centrifuged, the supernatant was discarded, and the cells were washed with PBS once, then the anti-mouse CTLA-4 antibody mCTLA-4 APC ( FIGs. 25A, 25B, 25C ) or the anti-human CTLA-4 antibody hCTLA-4 PE ( FIGs. 25D, 25E, 25F ), or the anti-mouse PD-1 antibody mPD-1 PE ( FIGs. 25G, 25H, 25I ) or the anti-human PD-1 antibody hPD-1 FITC ( FIGs.
  • T cells that express the human CTLA-4 and PD-1 proteins can be detected in the spleens of the gene humanized CTLA-4/PD-1 heterozygous mice for both the human-source CTLA-4 antibody and the human-source PD-1 antibody, while no T cells that express the human CTLA-4 or PD-1 protein were detected in the spleens of the C57BL/6 control mice.
  • the present disclosure provides a method for preparing a PD-1 gene-modified humanized mouse model.
  • the model utilizes the CRIPSR/Cas9 technique to replace partial fragments of a mouse PD-1 gene with fragments of a human PD-1 gene in a manner of DNA homologous recombination by constructing a targeting vector, thereby preparing a PD-1 gene-modified humanized mouse.
  • This mouse can normally express a PD-1 protein containing the functional domain of the human PD-1 protein, and can be used as an animal model for signal mechanism research regarding PD-1, PD-L1 and other genes and proteins, for screening individual or multiple effective regulators and drugs, and for pharmacological research.
  • the method has an important application value in studies on functions of the PD-1 and PD-L1 genes and in the development of new drugs.

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Claims (11)

  1. Souris humanisée à gène PD-1 modifié, dans laquelle le génome de la souris comprend une séquence d'acides nucléiques codant pour une protéine PD-1 chimérique comprenant la séquence d'acides aminés de SEQ ID n° : 17, et la souris exprimant la protéine PD-1 chimérique.
  2. Souris selon la revendication 1, dans laquelle le génome de la souris comprend la SEQ ID n° : 21, en option dans laquelle le génome de la souris comprend la SEQ ID n° : 14.
  3. Souris de la revendication 1, dans laquelle le gène PD-1 endogène est modifié par une protéine 9 (Cas 9) associée à CRISPR avec des ARNsg qui ciblent la SEQ ID n° : 3 et la SEQ ID n° : 8.
  4. Procédé destiné à préparer la souris humanisée à gène PD-1 modifié de l'une quelconque des revendications 1 et 2, comprenant les étapes suivantes :
    (1) fournir un plasmide comprenant un fragment de gène PD-1 humain qui est flanqué d'un bras d'homologie en 5' et d'un bras d'homologie en 3', dans lequel les bras d'homologie en 5' et en 3' ciblent l'exon 2 d'un gène PD-1 de souris, dans lequel le fragment de gène PD-1 humain comprend la SEQ ID n° : 21, dans lequel le bras d'homologie en 5' comprend la SEQ ID n° : 18 et le bras d'homologie en 3' comprend la SEQ ID n° : 24 ;
    (2) construire un plasmide d'ARNsg du deuxième exon du gène PD-1 de souris ;
    (3) injecter le produit de la transcription in vitro de l'étape (2), le plasmide de l'étape (1) et de l'ARNm de Cas9 dans un cytoplasme ou un noyau d'un ovule fécondé de souris C57BL/6 de type sauvage et transplanter l'ovule fécondé dans une souris femelle receveuse, dans le but de produire le modèle de souris humanisée à gène PD-1 modifié.
  5. Souris de l'une quelconque des revendications 1 et 2, dans laquelle le génome de la souris comprend en outre un deuxième gène humanisé.
  6. Souris de la revendication 5, dans laquelle le deuxième gène humanisé est un gène CTLA-4 humanisé.
  7. Cellule ou tissu isolé(e) à partir de la souris de l'une quelconque des revendications 1, 2 et 5.
  8. Procédé destiné à évaluer l'efficacité de substances médicamenteuses à cibles PD-1/PD-L1, dans lequel la souris selon l'une quelconque des revendications 1, 2 et 5, ou la cellule et le tissu selon la revendication 7, est/sont utilisé(e) (s) pour évaluer des substances médicamenteuses.
  9. Procédé destiné à évaluer l'efficacité d'une combinaison de substances médicamenteuses ciblant les PD-1/PD-L1 et d'autres substances médicamenteuses, dans lequel la souris selon l'une quelconque des revendications 1, 2 et 5, ou bien la cellule ou le tissu selon la revendication 7, est utilisé(e) pour évaluer des substances médicamenteuses ciblant les PD-1/PD-L1.
  10. Procédé destiné à cribler des substances médicamenteuses ciblant les PD-1/PD-L1, dans lequel la souris selon l'une quelconque des revendications 1, 2 et 5, ou bien la cellule ou le tissu selon la revendication 7, est utilisé(e) pour cribler des substances médicamenteuses.
  11. Procédé destiné à cribler une combinaison de substances médicamenteuses ciblant les PD-1/PD-L1 et d'autres substances médicamenteuses, dans lequel la souris selon l'une quelconque des revendications 1, 2 et 5, ou bien la cellule ou le tissu selon la revendication 7, est utilisé(e) pour cribler des substances médicamenteuses ciblant les PD-1/PD-L1.
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